The examination of the effect of biodiesel by-products and raw-materials on animals PhD - Thesis
Eszter Szele, MD
Doctoral School leader: Sámuel Komoly, MSc, DSc Program leader:
István Ember, MSc, DSc † István Kiss, MSc, DSc
Consultant:
István Ember, MSc, DSc † István Kiss, MSc, DSc
University of Pécs, Faculty of Medicine 2014.
I. Introduction I.1. Biofuels While the amount of fossil fuels is reducing, global energy demand is consistently increasing. Biofuels are alternative energy sources which may help to reduce air pollution, as well as our dependence on petroleum for energy. The so-called first generation biofuels refer to fuels that have been derived from food crops. The second generation biofuels, also known as advanced biofuels, are fuels that can be manufactured from various types of biomass such as the byproducts of agriculture, forestry and related industry. Sustainability criteria for biofuels and bioliquids are regulated by laws, protocols and action plans in the European Union and Hungary. In the European Union, the turn towards renewable energy sources has increased the production of biodiesel from rapeseed oil. I.2. Biodiesel production I.2.1. Biodiesel production from natural crops Biodiesel can be produced from a variety of natural crops including rapeseed, soybean, mustard, flax, sunflower, canola and palm oil. There are cultivatable agricultural fields in Hungary which are suitable for growing vegetable for biodiesel. Biodiesel is an ester based fuel for diesel engines produced by transesterification of short chain fatty acids of plant origin and mono alcohols, such as methanol. Biodiesel can be used on its own instead of fossil fuels or mixed with them. The transesterification reaction of the triglyceride from corn oil with an alcohol results in formation of esters of alcohols and glycerol. Glycerol is a natural liquid substance registered in the European Union as a feed additive, its code is E422. I.2.2. Biodiesel production from animal fat or used cooking oil Biodiesel can be produced from animal fat or used cooking oil as well. However, this process is more difficult and expensive than the production from natural crops. I.3. Experiments with biodiesel glycerol form natural vegetable oils For each litre of biodiesel, approximately 80-100 gram of crude glycerol is produced. Pure glycerol is colourless, odourless, nontoxic and it’s made of 3 carbon atoms, 8 hydrogen atoms and 3 oxygen atoms. Although glycerol in its pure form is used to manufacture soap, cosmetics, pharmaceuticals and other products, the biodiesel industry is producing more of this compound than the market can cope with. This has led to renewed efforts to find new ways for the utilisation of glycerine. One proposal is to use it as a pure energy source in animal diet. Several studies have evaluated the application of glycerol in diets for poultry, pigs and ruminants, and they found no detrimental effects on growth performance or meat quality. I.4. Experiments with biodiesel by-products or raw-materials of biodiesel I.4.1. Biodiesel glycerol One study, carried out at the University of West Hungary, Faculty of Agricultural and Food Sciences, investigated the metabolic effects of glycerol on boilers and barrows. Animals were fed 0, 5, 10 or 15% food grade (86,3 %) biodiesel glycerol, and they found that glycerol supplementation did not affect the quality and chemical composition of the meat, and the average daily weight did not differ between experimental and control animals. Szendi et al and Gerencsér et al from the University of Pécs studied the possible genotoxic effects of oral exposure of biodiesel glycerol. In a combined in vivo/ in vitro Ames test, Long
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Evans rats were feed with 2000 mg/kg biodiesel glycerol. Ames test was made from urine samples, while blood samples were taken for comet assay. They did not find any mutagenic or toxic effects of glycerol. I.4.2. Soapy water During purification of the crude biodiesel with slightly acidic warm water, a so-called soapy water fraction, containing glycerol, fatty acids, fatty acid methyl ester and various salts, is gained. The composition of the soapy water can provide important trace elements to the microorganisms in the soil, thus it has been suggested that it could be used to improve soil quality. Gerencsér et al studied the ecotoxicological effect of soapy water with white mustard (Sinapis alba) root growth assay. They mixed different concentration of soapy water with soil and examined the survival of worms in this soil. Ecotoxicological effect was not found. I.4.3. Corn oil and yellow grease We investigated the environmental effect of used corn oil (corn oil) and used cooking oil (yellow grease), because it has been suggested using it as biodiesel raw-material. Ames test was carried out by Szendi et al. Although they did not demonstrate any mutagenic activity of neither corn oil nor yellow grease, the white mustard root growth test suggested ecotoxicological effect of both. According to the results, the corn oil and the yellow grease are not suitable to improve soil quality, but their applicability as feed additive is not clear. I.5. Molecular epidemiologic experiments with biodiesel by-products and biodiesel rawmaterials To date, no studies investigated the carcinogenic effects of the by-products of biodiesel produced from vegetable oil and corn oil or yellow grease. One aim of the molecular epidemiological studies is to detect the cell damage with early biomarkers, which can be the tool of primer prevention, because they can indicate with modified expression the changed state even before the development of invasive tumours. I.5.1. Regulation of apoptosis The genes which regulate signalling pathway are also considered as early biomarkers. The altered expression of these genes suggested the effect of exposure on apoptosis or cell cycle. We aimed to analyse the effects of investigated samples on the expression of three genes which play crucial role in the regulation of apoptosis and cell death: the nuclear factor kappalight-chain-enhancer of activated B-cells 1 (Nfkb1), the growth arrest and DNA-damageinducible protein 45 alpha (Gadd45a) and the mitogen-activated protein kinase 8 (Mapk8, JNK1). I.5.2. Regulation of biotransformation The effect of exposure can be detected in the changed biotransformation. The enzymes that play a role in the metabolism of xenobiotics can be classified into two main groups. Phase I metabolic enzymes produce small chemical changes that make a compound more hydrophilic, so it can be effectively eliminated by the kidneys. For example, Cytochrome P450 enzymes are responsible for most phase I reactions. Phase II metabolic enzymes are activated if phase I is insufficient to clear a compound from circulation, or if phase I generates a reactive metabolite. The changes in the gene expression of cytochrome P450 enzymes can be a first sign of damaging effect. We analysed gene expressions of cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1) and cytochrome P450, family 2, subfamily e,
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polypeptide 1 (Cyp2e1). These genes encode two metabolizing enzymes responsible for the oxidative transformation. I.5.3. Oncogenes and tumour suppressor genes A proto-oncogene is a normal gene that can become an oncogene due to mutations or increased expression. Activated oncogenes can be responsible for the survival and proliferation of the cells which are designated for apoptosis. A tumour suppressor gene, or antioncogene, is a gene that protects a cell from progressing towards the direction of cancer. The oncogenes and tumour suppressor genes are also early biomarkers, as the examined classical oncogene: v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-ras). I.5.4. microRNAs Recently microRNAs (miRNA) have become the focus of molecular genomic research. There is increasing interest in the relationship between miRNA expression profile and exposure to carcinogenetic agents. They can function as oncogenes or tumour suppressors, and also have a role in immune response. The expression of miRNAs is associated with disease outcomes, postsurgical recurrences and even metastatic potential in cancer patients. miRNA expression profiles can have predictive value for assessing chemical carcinogenesis. We evaluated the expression of some tumour suppressor miRNAs (miR-34a, miR-143, miR146a, miR-203, miR-223), and some oncomirs (miR-21, miR-27a, miR-93, miR-148a, miR155, miR-196a, miR-205, miR-221). II. Objectives Testing the environmental effects of biodiesel raw materials and by-products is essential before recycling them as feed additive or using them to improve soil quality. Our aim is to examine the corn-based biodiesel by-products, the corn oil and the yellow grease, and study their effect on carcinogenesis. II.1. The effect of biodiesel glycerol Our objective is the investigation of environmental health risk of biodiesel glycerol (with different concentration) on apoptosis, biotransformation, K-ras oncogene and several miRNAs. II.2. The effect of soapy water Our objective is investigation of environmental health risk of wheat growing on soil treated with different concentration soapy water on apoptosis, biotransformation and several miRNAs. II.3. The effect of corn oil and yellow grease Our objective is the investigation of environmental health risk of corn oil and yellow grease on apoptosis, K-ras oncogene and several miRNAs. III. Materials and methods III.1. Research animals Our research was funded by a tender and the investigations were carried out as part of a consortium. The consortium aims to produce biodiesel by-products which can be recycled safely. Our institute was responsible for carrying out the project on environmental health inspections. Experiments were carried out according to Hungarian laws, and the Code of Ethics and Animal Research of University of Pécs. In the experimental cancer research particular inbred rodents were used which have spontaneous incidence of tumours. A short-term animal test system has been developed in our institute, which indicated in a very early
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stage of the carcinogenic effect of chemical carcinogens. Three different inbred mouse types were avaiable during the research period: CBA/Ca, BALB/c and AKR/J. Each mouse type shows elevated susceptibility for tumour formation, they are equally sensitive for various carcinogenic effects. We determined the gene expression in each kind of mice to determine the difference between the results. III.2. Investigated samples The biodiesel glycerol and soapy water was produced by KUKK K+F Ltd. (Budapest), the standard chew pellet by Szinbád Ltd. (Gödöllő), corn oil and yellow grease by QS Biodízel Ltd. (Newton, USA), its contains: • standard chew pellet: energy: 11 MJ/kg, dry matters: 86%, crude protein: 20%, enzyme protein: 18,2%, lysine: 0,97%, methionine: 0,30%, cysteine: 0,64%, crude fat: 4%, crude fibre: 4,30%, Ca: 1,08%, P: 0,85%, Na: 0,20%, Vitamin A: 18000 NE/kg, Vitamin D: 1000 NE/kg, Vitamin E: 75 mg/kg • low purified biodiesel glycerol: glycerol: 60%, vegetable oil: 20%, P: 4%, Na: 1%, K: 5%, methanol: 0,04%, water and other mineral components: 9,96% • low purified biodiesel glycerol: glycerol: 86,3%, vegetable oil: 5%, P: 2%, Na: 1%, K: 2%, methanol: 0,04%, water and other mineral components: 5% • soapy water: methanol: 23, 3 m/m%, glycerol < 0,06 m/m %, Cl: 36,7 mg/l, P: 71,7 mg/l, S: 38,5 mg/l, nitrite: 0,3 mg/l, nitrate: 29,2 mg/l, fatty acids < 0,1 m/m% • the composition of corn oil and yellow grease, which compounds are used as biodiesel raw materials, constantly changes. III.3. Exposure Mice received humane care and the experiment was carried out under the approval of the Institutional Revision Board. Mice were 6 weeks old, between 20-25 grams. Mice were fed with 10% purified biodiesel glycerol, corn oil and yellow grease. Wheat, which was growing on soil treated with different concentrations of soapy water, was given in 100% to mice. Control animals consumed standard chew pallet. The daily food intake was 3 grams. After administration, animals were sacrificed by cervical dislocation and the examined tissues of the animals were removed during autopsy. The tissues were homogenized and pooled by group. Subsequently, total cellular RNA was isolated from the tissues with MagNA Pure Compact automatic nucleic acid isolation system (Roche, Berlin, Germany) according to the manufacturer's instructions. The quality of the isolated RNA was checked by absorption photometry at 260/280 nm. Optical density of the RNA was between 1.9 and 2.1. III.4. Experiment groups (A1, A2, A3, B, C) The experiments were maintained in three main groups: A, B and C, and there were three sub-group in the A main group: A1, A2, A3. The details are shown in Table I., as inbreed mouse type, exposition material and time, the investigated genes. III.5. Mesaure of gene expression We used the kits and instrument of Roche (Berlin, Germany) during the experiments. Total cellular RNA was isolated from the homogenized tissues with High Pure miRNA Isolation Kit according to the manufacturer's instructions. The concentration of RNA was detected by absorption photometry. Total RNA was reverse transcribed, then PCR reactions were carried out. Primers were selected by the primer finder database (www.applied-science.roche.com) and were synthesized by TIB Molbiol, ADR Logistics. The gene expressions of mRNAs were calculated relative to the expression of Hprt, miRNAs expression to 5S rRNA. Statistical evaluation was carried out by paired t-test using STATA Release 11 software for Windows (StataCorp LP, Texas, USA). Values of p<0.05 were considered to be statistically significant. III.6. Statistical probes All PCR reactions were run in triplicates in separate runs. The concentration of miRNAs and mRNAs were determined in tissues and averaged. Statistical evaluation was carried out by paired t-test using STATA Release 11 software for Windows (StataCorp LP, Texas, USA). Values of p<0.05 were considered to be statistically significant. Some investigator defined that miRNA expression is elevated twice or even tenfold in tumour tissue compare to normal. In order to avoid false positive results, we defined that miRNAs expression changes have to be elevated at least three times to determine significant impact in the carcinogenesis.
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Table I. Experimental groups
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IV. Results IV.1. Biodiesel glycerol IV.1.1. Effect of biodiesel glycerol on apoptosis (A1 experiment) Expressions of the investigated genes showed clearly higher gene expressions after administration of biodiesel glycerol. (Figure 1.)
2,5
Nfkb1 (female)
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Nfkb1 (male)
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: biodiesel glycerol (60%)
: biodiesel glycerol (86,3%) *: p< 0,05
Figure 1: Gene expression of Nfkb1 and Gadd45a in different tissues of female and male mice proportion to Hprt internal control after 3, 6 or 24 hours of biodiesel glycerol diet compared to controls
Lower purified biodiesel glycerol reduced Nfkb1 expression nearly half of the control expression, and this downregulation of Nfkb1 expression was seen in both males and females in every exposure times. Lower purified biodiesel glycerol consumption in male mice resulted in grater under expression than the females in all investigated organs, and the expression suppressions ranged between 47-75% of the control values at all-time points. Gadd45a expression showed negligible changes both in the liver and the spleen of female mice after administration. In the liver of male mice Gadd45a expression altered similarly to Nfkb1 expression in all three time points. After higher purified biodiesel glycerol administration, Nfkb1 expression values were lower in female’s liver after 3 and 6 hours of consumption, in liver after 24 hours of consumption, and Gadd45a expression reduced significantly in bone marrow and liver. However, in male groups Nfkb1 expressions of the spleen and the bone marrow were higher in the 24 hour time point, surpassing the level of the control's 57% in the spleen and 82% in the bone marrow. Gadd45a was suppressed in the spleen and in the bone marrow of male mice after 3 and 6 hours of administration, and was elevated in liver at every time point.
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Based on results obtained in the A1 experiment, we didn’t continue the investigation with lower purified biodiesel glycerol, because its harmful effect could not be excluded. In the following parts of the thesis, if not marked otherwise, the biodiesel glycerol means higher purified glycerol. The change in gene expression was the most marked in the liver. Furthermore liver plays a key role in the metabolism of methanol in the body, so we continued our experiments only in liver. IV.1.2. Effect of biodiesel glycerol on biotransformation (A2 experiment) According to our results, the expression of Cyp1a1 and Cyp2e1 in liver was significantly higher at the 3hour time point in both genders. Cyp2e1 overexpression was more marked in both sexes, reaching a four-fold overexpression in female mice and a three-fold in the males. At the 6-hour time point Cyp1a1 expression returned to the control's level, while Cyp2e1 tended to decrease but remained significantly up-regulated in female mice, reaching a nonsignificant level in males. Figure 2. shows that the up-regulation of the genes returned to the expression levels of the controls within 24 hours from administration. Cyp1a1 15
male
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*: p< 0,05
Figure 2: Gene expression of Cyp1a1 and Cyp2e1 in liver of female and male mice proportion to Hprt internal control after 3, 6 or 24 hours of biodiesel gycerol diet, compared to controls
The change in gene expression was the most marked after 24 hours administration, so we continued our experiment only with 24 hours exposure. IV.1.3. Effect of biodiesel glycerol on K-ras oncogene, oncomirs and tumour suppressor miRNAs and mRNAs expression (A3 experiment) Male mice exhibited more significant expression changes in the investigated miRNAs than females. In the group of female mice, the oncogene miR-27a was significantly deregulated parallel to down-regulation of oncogene K-ras and antiapoptotic Nfkb1 and Mapk8, while two other oncogene miRNAs, miR-34a and miR-221 were found to be overexpressed. In male mice 24-hours’ biodiesel glycerol administration resulted in the up-regulation of oncogene miR-93a and miR-221, while the also oncogene miR-155 with miR-196a were observed to be down-regulated by the under-expression of K-ras. In male mice Nfkb1 expression did not alter, while Mapk8 was also down-regulated. The investigated mRNA exhibited significant expression change in all examined animals and almost in all examined genes, while expression of oncogene miR-21, miR-143, miR-148a and miR-205 and tumour suppressor miR-146a in animals was similar to control.
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The expression alteration reached the three-fold level only in few cases. The results are show in Figure 3. 500
miR-21 female
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Figure 3: Gene expression of miRNAs compared to 5S RNA internal control, and Nfkb1, Mapk8, K-ras genes proportion to Hprt internal control in liver of female and male mice after 24 hours of biodiesel glycerol diet, compared to controls
IV.2. Effect of wheat, grown in soapy water treated soil, on apoptosis, biotransformation, oncomirs, tumour suppressor miRNAs and K-ras oncogene (B experiment) We compared the effect of wheat grown in soapy water treated soil to wheat grown in normal soil. Wheat grown in soil treated with 1000 l/ha soapy water showed significantly overexpression of a miR-21 and significantly downregulation of miR-146a in male mice, but these changes
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didn’t reach the 3 time level. The level of miR-221 was more than three times higher in every gender compared to controls. Wheat grown in soil treated with 500 l/ha soapy water supressed the expression of miR-221 with 60%. There were no significant changes in the expression of miRNAs after administration of wheat grown in soil treated with 250 l/ha soapy water. Nfkb1 expression was significantly higher, while Mapk8 was significantly lower after 1000 l/ha and 500 l/ha soapy water treatment in both gender, and Nfkb1 was also overexpressed in female mice in the case of 250 l/ha concentration. The soapy water had effect on biotransformation only in 1000 l/ha concentration. Cyp1a1 was under expressed in female and Cyp2e1 in male mice. (Figure 4.) miR-21
miR-27a male
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Figure 4: Gene expression of miRNAs compared to 5S RNA internal control, and Nfkb1, Gadd45a, Mapk8, Cyp1a1, Cyp2e1 genes proportion to Hprt internal control in liver of female and male mice after 24 hours diet with wheat growing on soil treated with soapy water in different concentration, compared to controls
IV.3. Effect of corn oil and yellow grease on apoptosis, on oncomirs and tumour suppressor miRNAs and K-ras oncogene (C experiment) Expression of the investigated genes showed many difference between controls and mice consumed corn oil or yellow grease. (Figure 5.) Corn oil administration reduced the expression of miR-21, miR-27a, miR-93, miR-143, miR146a, miR-155, miR-203 and miR-221 level in female mice, reduced one-third of the miR-27a
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and the miR-146a. In male liver, suppressed expression of miR-34a, miR-93, miR-143, miR146a, miR-155, miR-196a, miR-203 and miR-221 were showed. From these, only the miR146a was reduced by third, and the miR-27a was overexpressed. The Mapk8 expression was lower in female mice, while Nfkb1 and K-ras in both genders compared to controls. After administration of yellow grease, miR-21, miR-27a, miR-34a, miR-93, miR-155 and miR221 were downregulated in both genders. The miR-143, miR-146a and the miR-205 were suppressed in females, miR-148a and miR-196a in males. Two miRNAs were upregulated in male mice: miR-146a and miR-203. Nfkb1 and K-ras was overexpressed in males.
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Figure 5: Gene expression of miRNAs compared to 5S RNA internal control, and Nfkb1, Mapk8, K-ras genes proportion to Hprt internal control in liver of female and male mice after 24 hours diet with corn oil or yellow grease, compared to controls
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V. Discussion The bioutilization and biological effect of biodiesel by-products in vivo strongly depend on the impact of trace elements, saturated and unsaturated fatty acid and methanol content of the product. Our aim was to develop the technology for biodiesel glycerol purification, in order to gain a glycerol fraction that could be physiologically optimal for animal feeding, with the cost effectively highest purity and with the lowest possible risk of carcinogenicity. It would be more cost-effective and environmental friendly to use the by-product of a renewable energy product. The corn oil and yellow grease can be used for biodiesel production, and we investigated whether they are suitable as food additive too. V.1. Regulation of apoptosis NFkB is a protein complex that acts as a transcription factor and regulates genes that control cell proliferation and cell survival and mediate the inflammatory response. The NFkB family includes homo- and heterodimers that are formed from five structure-related protein subunits: p50 (NFkB1), p52 (NFkB2), p65 (RelA), c-Rel, Rel B. NFkB can be found in various cells. NFkB normally binds to IkB inhibitors. Stress stimulates IkB kinase, and activated IkB kinase mediates IkB phosphorylation. The NFkB-IkB complex degrades and the free NFkB translocates into the nucleus and regulates the transcription of target genes. NFkB complex can activate different pathway, the canonical (alternative) and the non-canonical (alternate) pathway, and so has apoptotic or antiapoptotic effects depending on the manner of its induction. Gadd45a is a member of a group of genes whose transcript levels are increased following stressful growth arrest conditions and treatment with DNA-damaging agents. Gadd45 family members interact with the upstream kinase mitogen-activated protein kinase 4 (Mekk4) which activates JNK. The down-regulation of Gadd45a leads to NfkB-dependent escape from programmed cell death through the JNK cascade. The arsenite-induced AP-1 activation was delivered by sequential induction of GADD45a expression and the activation of MAPKK (MKK3/4/6) and MAPK (JNKs and p38K)dependent pathways. V.1.1. Effect of biodiesel glycerol on apoptosis Administration of lower purified biodiesel glycerol caused downregulation of Nfkb1 and Gadd45a in almost every tissue, in every time point and in both genders. This downregulations reached the remarkable 50% in the bone marrow of both sexes. Based upon this finding, we can conclude that implication of biodiesel glycerol in the diet results in early gene expression downregulation on the extrinsic apoptotic cascade via suppression on the DNA damage inducible Gadd45a expression and suppression of Nfkb1, potentiating cell survival mechanisms of the cells with damaged genetic material. Therefore, our workgroup though that it is not safe to use it as a feed composition. Higher purified biodiesel glycerol had much smaller effect on gene expressions of the vast of the investigated organs, although isolated induction of Gadd45a were seen in the spleen and bone marrow of male mice in the 24 hour time point and in liver in every time point. Nfkb1 expression was lower in female BALB/c mice, and higher in male CBA/Ca mice compared to the control, and the Mapk8 was downregulated in BALB/c mice. By the alteration of Nfkb1 and Mapk8 gene, it can be apoptotic or antiapoptotic too, but the upregulated Gadd45a even suggested antiapoptotic effect. These data suggest that glycerol fractions with higher purity has no harmful effect on the apoptotic signalling, thus on the evasion of apoptosis and cell survival.
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V.1.2. Effect of wheat growing on soil treated with soapy water on apoptosis Wheat growing on soil treated with soapy water caused downregulated Mapk8 and upregultaed Nfkb1 levels, and also the expression was elevated of Gadd45a, but it was only significant in female mice. According to Gadd45a upregulation and the inverse expression of Nfkb1 and Mapk8, it seems to have apoptotic effect. Our result suggests that soapy water has no harmful effect on apoptosis. V.1.3. Effect of corn oil and yellow grease on apoptosis According to our result with corn oil, the expression of Nfkb1 was elevated in both gender, and Mapk8 was upregulated in female liver. The yellow grease had effect only on Nfkb1 gene expression, which was elevated in male. Both corn oil and yellow grease have effect on investigated genes in males, but yellow grease has effect only on males. Our results suggest that these materials influence the expression of genes, which play central role in cell survive. V.2. Regulation of apoptosis Cyp1a1 encodes a protein which is localised in the endoplasmic reticulum. Endogenous substrates of Cyp1a1 are steroids and fatty acids, and it also partakes in metabolism of caffeine, pethidine, phenacetin, progesterone and other steroids and polycyclic aromatic hydrocarbons. Cyp2e1 is also localised in the endoplasmic reticulum, and it is involved in the metabolism of endogenous substrates such as acetone and acetol, as well as exogenous substrates, for example acetaminophen, halothane, isoflurane, paracetamol, benzene, aniline, nitrosamines, ethanol and methanol. In addition, in rodents, alcohol dehydrogenisation is also maintained mostly by Cyp2e1 and less by the alcohol dehydrogenases. Levels of Cyp2e1 are elevated under a variety of physiological and pathophysiological conditions, such as alcohol and xenobiotic exposure. Cyp2e1 is an effective generator of ROS, such as the superoxide anion radical and hydrogen peroxide, and in the presence of iron catalysts, it produces powerful oxidants such as the hydroxyl radical. Lu et al. demonstrated on in vivo rodent liver models that Cyp2e1-derived oxidative stress may inhibit oxidation of fatty acids, resulting in steatotic hepatic lesions. V.2.1. Effect of biodiesel glycerol on biotransformation According to our results, the expressions of both Cyp1a1 and the Cyp2e1 were elevated after supplementing the animals' diet with biodiesel glycerol, as a response to the exposure due to its variety of lipid contaminants and methanol. This upregulation quickly subsided and expressions equilibrated with those of the controls within six hours, suggesting that the pathway of biotransformation adapted to the metabolic needs. No significant alterations of the genes were seen after 6 and 24 hours of exposure. Sex differences in expression could occur due to the fact that the microsomal density of CYP2E1 in female mice is higher as activity of microsomal mono-oxygenize activity is modified by sex hormones. Based upon our data, the higher purified biodiesel glycerol seems to have only a transient short-term effect on these two genes, which is lost within 24 hours, putting a minimal and reversible exposure burden on the metabolic balance of the animals. V.2.2. Effect of wheat growing on soil treated with soapy water on biotransformation The wheat which was grown in soil treated with 1000 l/ha soapy water had effect only on cytochromes. The expression of Cyp1a1 was decreased in female mice, while the expression of Cyp2e1 was lower in the liver of male mice. The lower concentration of soapy water had no effect on biotransformation, but it is possible, that methanol can cause changes in 1000
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l/ha concentration, so we suggest to avoid using it for soil quality improvement in this concentration. V.3. Effects on K-ras oncogene Rat sarcoma virus oncogene (RAS) proteins were originally identified as retroviral oncogenes, they are small GTPases. RAS proteins control cellular signalling pathways responsible for growth, migration, adhesion, cytoskeletal integrity, survival and differentiation. As many human tumours have activating mutations in one of the RAS genes, RAS signalling may result in malignant transformation. V.3.1. Effects of biodiesel glycerol on K-ras oncogene After exposure with higher purified biodiesel glycerol, the expression of K-ras was lower than in controls, therefore biodiesel glycerol seems to be a protective agent. V.3.2. Effects of corn oil and yellow grease on K-ras oncogene Administration of corn oil elevated K-ras expression in both genders. It was overexpressed in male’s liver after diet with yellow grease. Our results suggest that corn oil and yellow grease has carcinogenetic effect, therefore they should not be used as feed additive or for soil quality improvement. V.4. Effect on oncomirs and tumour suppressor miRNAs Several studies have shown that miRNAs regulate cell proliferation and apoptosis, they function as oncogenes or tumour suppressors and also have a role in immune response. miRNA expression profiles can have predictive value for assessing chemical carcinogenesis. The miRNA profile can be different in alcoholic and non-alcoholic steatosis. The liver contains many types of cells, including parenchymal cells (i.e. hepatocytes) and “nonparenchymal cells” which can be endothelial cells, stellate cells, lymphoid cells, and biliary epithelial cells (cholangiocytes). Each cell type may have completely distinct miRNA expression profile because most of the cells are hepatocytes and the homogenized tissue expression profile is typifying hepatocytes. The oncomirs are the follows: miR-21, miR-27a, miR-93, miR-148a, miR-155, miR-196a, miR-205, miR-221. miR-21 has multiple functions: it promotes cell proliferation, migration, survival and also promotes cyclin D1 translation; it helps hepatocytes to exit from the G0 phase of the cell cycle to progress through the G1 phase; it also inhibits the negative regulators of the Ras/MEK/ERK pathway and inhibit apoptosis. The increased expression of the miR-23a-27a-24 cluster decreased the level of the tumour suppressor transforming growth factor-beta (Tgf-b) in hepatocellular carcinomas. miR-27a may modulate expression of miR-27a a zinc finger and BTB domain containing 10 (Zbtb10) and myelin transcription factor 1 (Myt-1) tumour suppressor genes, that inhibit cells from progressing past G2-M phase. miR-93 promotes tumour growth and angiogenesis by suppressing integrin-β8 expression and inhibited Fus1 protein expression. Elevated expression of miR-148a can be detected in aggressive tumour tissues, it inactivates the tumour suppressor phosphatase and tensin homolog (PTEN). The expression of miR-155 was significantly higher in the liver tissue of mice after a choline-rich diet. Choline presence indicates an inflammatory response, and exposure via animal diet causes hepatocellular carcinoma. miR196a promoted the epithelial-mesenchymal transition and migration/invasion capabilities of transfected cells, suggesting its oncogenic potential, and promotes cell proliferation by downregulating p27. miRNA-221 in tumour samples target the CDK inhibitor p27 and enhance cell growth in vitro, and miR-221 overexpression stimulates growth of tumourigenic murine hepatic progenitor cells. 14
The tumour suppressors are: miR-34a, miR-143, miR-146a, miR-203 and miR-223. The miR-34a, as a tumour suppressor, suppresses tumour migration and invasion through modulation of the c-Met signalling pathway. KRAS oncogene has been further experimentally validated as the target of miR-143, there is an inverse correlation between KRAS protein and miR-143. Expression of miR-146a suppresses NF-kB activity and reduces the metastatic potential of tumour cells. Functional polymorphism in the miR-146a gene is associated with the risk for hepatocellular carcinoma. miR-203 was found to be tumour suppressor miRNA in hepatocellular carcinoma. The ATP-binding cassette, sub-family E (OABP), member 1 (ABCE1) is a possible target for miR-203. ABCE1 is known to bind with eukaryotic initiation factors and play a role in vertebrate translation initiation. ABCE1 inhibitors were shown to efficiently suppress the growth of human tumour cells. One of the targets of miR-205 is the Kras oncogene. In hepatocellular carcinoma cells miR-223 was repressed compared with normal liver tissue. It seems to inhibit insulin-like growth factor-1 receptor (IGF-1R) signal pathway, as a tumour supressor gene. V.4.1. Effect of biodiesel glycerol on miRNAs expression Biodiesel glycerol diet had no remarkable effect on the expression of miRNAs. The elevation of their expression did not reach the three-fold level and their quantity did not decrease to its third.. V.4.2. Effect of wheat grown in soapy water treated soil on miRNAs expression Consumption of wheat grown on soil treated with 1000 l/ha soapy water dowregulated the expression of miR-221 in both genders by one- third, but it had no more remarkable effect. V.4.3. Effect of corn oil and yellow grease on miRNAs expression The level of oncomir miR-27a in females, and the level of tumour suppressor miR-146a in males decreased to its third after corn oil diet. After administration of yellow grease, the tumour suppressor miR-146a decreased more than a third in female mice’s liver while in male mice increased more than fourfold. The expression of oncogenic miR-148a decreased to its eighth in males. Carcinogenic effect was not confirmed on the basis of differences in miRNA expression in either corn oil or consumption of yellow grease enriched diet. VI. Summary and the new findings Biofuel companies can produce biodiesel in principally from vegetable oil, but due to the economic requirements they try to produce it from used cooking oil and animal fats. The rising production of rapeseed evoked the necessity of the utilization of the valuable byproducts, the glycerol fractions. At the Institute of Public Health at the University of Pécs we examined the alteration in gene expression profiles in animals after administration of biodiesel by-products and raw materials. We studied their effect on carcinogenesis and found that they could be physiologically optimal for animal feeding, or for using them as soil quality improving compounds, before they enter into the food chain. VI.1. New findings • According to the expression changes of examined genes, the lower purified biodiesel glycerol, may have antiapoptotic effect. • According to the expression changes of examined genes, the higher purified biodiesel glycerol has no effect on apoptosis and has a transient effect on biotransformation. As it
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• • • • • •
suppressed the expression of oncogene K-ras, it can also be a protective agent. It has no remarkable effect on the examined miRNAs. According to the expression changes of examined genes, the wheat which was grown in soil treated with 1000 l/ha soapy water has apoptotic and metabolic effect, and can be protective agents as well, as it suppressed the expression of oncogene. According to the expression changes of examined genes, the wheat which was grown in soil treated with 500 l/ha soapy water, has apoptotic effect, has no effect on biotransformation and on examined miRNAs. According to the expression changes of examined genes, the wheat grown on soil treated with 250 l/ha soapy water, has apoptotic effect, has no effect on biotransformation and on examined miRNAs. According to the expression changes of examined genes, the wheat grown on soil treated with 125 l/ha soapy water has no effect on apoptosis, on biotransformation and on examined miRNAs. According to the expression changes of examined genes, corn oil has apoptotic effect, can be carcinogen, because it elevated the expression of K-ras oncogene, and has different effect on examined miRNAs. According to the expression changes of examined genes, yellow grease has apoptotic effect in female mice, and has no apoptotic effect in males. It can be carcinogen because it elevated the expression of K-ras oncogene. It also has different effect on examined miRNAs.
Based upon our data, the higher purified biodiesel glycerol can be used as feed additive and the soapy water which is less than 500 l/ha concentration is a suitable compound to improve soil quality. They pose no risk for the environment. The utilisation of corn oil and yellow grease as raw material for biodiesel production increases the cost-effectiveness of this process, because their recycling into the food chain is not safe as long as they have carcinogenic effect.
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VII. Publications The thesis is based on the following international publications 1. Szele E, Gombos K, Kovács A, Ember I: Feeding purificated glycerol from biodiesel to CBA/CA mice: effects on Gadd45α and Nfκb1 expressions. In Vivo 24(3): 303-307, 2010. imp.f.: 1,159 2. Szele E, Gombos K, Kovács A, Ember I: Effects of purified glycerol from biodiesel on Cyp1a1 and Cyp2e1 expressions in CBA/CA mice. In Vivo 25(2): 237-240, 2011. imp.f.: 1,264 3. Szele E, Gombos K, Juhász K, Wohler V, Kovács A, Ember I: Effects of purified glycerol from biodiesel on miRNAs compared to the expression profile of selected mRNAs in BALB/c mice. In Vivo 27(1): 107111, 2013. imp. f.: 1,264 4. Kádár B, Gombos K, Szele E, Beregi A, Varga Zs, Sebestyén A, Ember I: Effects of Isoflurane Exposure on Oncogene and Tumour Suppressor Gene Expressions in Vital Orga ns of CBA/CA Mice. In Vivo 21(5): 861-865, 2007. imp. f.: 1,143 5. Szanyi I, Lujber L, Gerlinger I, Pytel J, Bauer M, Csejtei A, Szele E, Gombos K, Kiss I, Seredenin S, Yarkova M, Ember I: In vivo effects of Afobazole (2-Mercaptobenzimidazole Derivate) on the 7,12Dimethylbenz [a] anthracene-induced oncogene and suppressor gene expression. In Vivo 21(6): 10591063, 2007. imp. f.: 1,143 6. Gombos K, Szele E, Kiss I, Varjas T, Puskás L, Kozma L, Juhász F, Kovács E, Szanyi I, Ember I: Characterization of microarray gene expression profiles of early stage thyroid tumours. Cancer Genomics and Proteomics 4(6): 403-409, 2007. 7. Gőbel GY, Gombos K, Szele E, Kálmán E, Budán F, Gerlinger I, Fiscina F, Szanyi I, Ember Á, Németh Á, Ember I: Retrospective analysis of malignant salivary gland tumors in Hungarian population between 19872006. European Journal of Oncology, 14(4): 209-215, 2009. imp.f.: 0,325 8. Kádár B, Gombos K, Szele E, Ember I, Iványi JL, Csejtei A, Pajkos G: Effects of Isoflurane on Nfκb p65 , Gadd45a and Jnk1 expression in the vital organs of CBA/CA mice. In Vivo 25(2): 241- 244, 2011. imp.f.: 1,264 The thesis is based on the following Hungarian publications 1. Szele E, Gombos K, Ember I: Biodízel előállítás során a glicerin-fázis melléktermékek állati takarmánykompozícióként való alkalmazásának vizsgálata – Az SZME2 hatása az NFKB1 és GADD45α expresszióra. Magyar Epidemiológia 6(1): 21-26, 2009. 2. Szele E, Gombos K, Juhász K, Wohler V, Kovács A, Ember I: Biodízel előállításra felhasznált kukoricaolaj és sárgazsír karcinogenezisben betöltött szerepének állatkísérletes vizsgálata különböző mRNS-ek és miRNS-ek kifejeződésének mérésével. Magyar Epidemiológia 9(3): 173-182, 2012. 3. Szele E, Gombos K, Juhász K, Kovács A, Ember I: Biodízelgyártás során visszamaradt szappanos vízzel kezelt talajon termesztett búza metabolizmusra és karcinogenezisre gyakorolt hatásának vizsgálata állatkísérletes modellben. Magyar Epidemiológia 9(3): 183-192, 2012. 4. Kádár B, Gombos K, Szele E, Gőbel Gy, Szanyi I, Ember I: Az Isoflurane in vivo hatástani vizsgálata. Magyar Epidemiológia 5(3-4): 181-190, 2008. 5. Ember I, Gombos K, Prantner I, Szele E: A betegségmegelőzés általános alapjai. Népegészségügyi orvostan (szerk.: Ember I.) Dialóg Campus, Pécs, 2007. 6. Ember I, Gombos K, Szele E: Genetika/genomika a népegészségügyben, genomikai epidemiológia. Népegészségügyi orvostan (szerk.: Ember I.) Dialóg Campus, Pécs, 2007. 7. Fehér K, Prantner I, Szele E, Németh Á, Berényi K, Huszár A, Iványi JL, Csejtei A, Sebestyén A, Ember I: A rosszindulatú daganatok megelőzése-prevenciós modell a háziorvostól a molekuláris epidemiológiáig. Magyar Epidemiológia 8(3): 145-161, 2011. 8. Gőcze K, Marek E, Gombos K, Prantner I, Szele E és Ember I: A betegségmegelőzés általános alapjai. Népegészségügyi Orvostan 2. kiadás (szerk.: Ember I, Kiss I, Cseh K; Dialóg Campus): 125-127, 2013. 9. Kiss I, Orsós Zs, Gombos K, Prantner I, Szele E, Ember I: Szűrés, szűrővizsgálatok. Népegészségügyi Orvostan 2. kiadás (szerk.: Ember I, Kiss I, Cseh K; Dialóg Campus): 128-132, 2013. Citeable abstracts: 1. Szele E, Gombos K, Kovács A, Ember I: Feeding purificated glycerol from Biodiesel to CBA/CA mice: effects on single strand DNA damage inducible GADD45α and NFKB expressions. Anticancer Research 28: 3297, 2008. imp. f: 1,39 2. Gombos K, Szele E, Puskás L,Kozma L, Juhász F, Gőbel Gy, Szanyi I, Ember I: Analysis of the connections between signal transduction mechanisms in early stage thyroid tumours. Anticancer Research 28: 3296, 2008. imp. f: 1,39 3. Kádár B, Gombos K, Szele E, Gőbel Gy, Szanyi I, Ember I: Effects of Isoflurane on NFKB1, GADD45α JNK1 expressions in the vital organs of CBA/CA mice. Anticancer Research 28: 3296, 2008. imp. f: 1,39 Citeable Hungarian abstracts:
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Szele E, Gombos K, Varjas T, Puskás L, Kozma L, Juhász F, Ember I: Microarray módszer alkalmazása a pajzsmirigy daganatok korai felismerésében. Magyar Epidemiológia Supplementum 5: 95-96, 2008. 2. Szele E, Gombos K, Ember I: Az SZME2 biodízel előállítás során nyert tisztított glicerin frakció NFKB, JNK és GADD45A génekre gyakorolt hatásának vizsgálata állatkísérletes modellben. Magyar Epidemiológia Supplementum 5(2): 174, 2008 3. Szele E, Gombos K, Ember I: Biodízel előállítása során nyert tisztított glicerin frakció apoptikus génekre gyakorolt hatásának vizsgálata állatkísérletes modellben. Magyar Epidemiológia Supplementum 6: 104, 2009. 4. Gombos K, Szele E, Herceg M, Brunner Zs, Szanyi I, Molnár K, Gergely P, Mucsi Gy, Varga Zs, Ember I: A VitaCalen® krónikus fogyasztása során észlelt eredményeink állatkísérletes modellben. Magyar Epidemiológia Supplementum 3: 41, 2006. 5. Gombos K, Szele E, Varjas T, Tetinger A, Molnár K, Varga Zs, Sebestyén A, Tibold A, Ember I: A VitaCalen® nevű étrendkiegészítő hatása onko- és tumorszuppresszor gén expressziókra. Magyar Epidemiológia Supplementum 3: 42, 2006. 6. Gombos K, Szele E, Varjas T, Puskás L, Kozma L, Juhász F, Ember I: Jelátviteli mechanizmusok összefüggéseinek vizsgálata pajzsmirigy daganatokban. Magyar Epidemiológia Supplementum 5: 44, 2008. 7. Kádár B, Gombos K, Szele E, Beregi A, Varga Zs, Sebestyén A, Ember I: Az izoflurán onko- és tumorszuppresszor génekre kifejtett hatásának vizsgálata CBA/Ca egerekben. Magyar Epidemiológia Supplementum 5: 55, 2008. 8. Kádár B, Gombos K, Szele E, Gőbel Gy, Szanyi I, Ember I: Az Isoflurane hatása az NFKB1, JNK1 és GADD45α gének expressziós mintázatára. Magyar Epidemiológia Supplementum 5(2): 150, 2008. 9. Gőbel Gy, Gerlinger I, Pytel J, Szanyi I, Szele E, Gombos K, Ember I: A Malignus nyálmirigy daganatok retrospektív vizsgálata az 1986-2006-os időtartamban. Magyar Epidemiológia Supplementum 5(2): 145, 2008 . 10. Gombos K, Szele E, Gőbel Gy, Puskás L, Kozma L, Juhász F, Ember I: Pajzsmirigy daganatok génexpressziós profiljának meghatározása cDNS microarray módszerrel. Magyar Epidemiológia Supplementum 6: 42-43, 2009. 11. Gőbel Gy, Gerlinger I, Pytel J, Szanyi I, Szele E, Gombos K, Ember I: Malignus nyálmirigy daganatok epidemiológiai sajátosságai. Magyar Epidemiológia Supplementum 6: 44, 2009. 12. Kádár B, Gombos K, Szele E, Gőbel Gy, Szanyi I, Ember I: Az Isoflurane hatása apoptikus jelátviteli gének expressziójára. Magyar Epidemiológia Supplementum 6: 53-54, 2009. International presentations: 1. Szele E, Gombos K, Ember I: Effects of biodiesel glycerol on DNA damage inducible and apoptotic genes. Népegészségügyi Tudományos Társaság XVII. Nemzetközi Kongresszusa, 2009. április 17-18., Marosvásárhely 2. Szele E, Gombos K, Ember I: Effects of Purified Glycerol from Biodiesel on Cyp1a1 and Cyp2e1 Expressions in CBA/CA Mice. International Conference of Preventive Medicine and Public Health, Pécs, 2010. november 19-20., Pécs 3. Szele E, Gombos K, Ember I: 2011. április: Feeding Animals with Biodiesel Glycerol. Effect of Biodiesel G- fractions on Genes Regulating Microsomal Metabolism. BIT’s 1st Annual World Congress of Bioenergy, 2011. április 25-29., Dalain, China 4. Gombos K, Szele E, Gőbel Gy, Puskás L, Kozma L, Juhász F, Ember I: Pajzsmirigy daganatok génexpressziós profiljának meghatározása cDNS microarray módszerrel. Népegészségügyi Tudományos Társaság XVII. Nemzetközi Kongresszusa, 2009. április 17-18., Marosvásárhely 5. Kádár B, Gombos K, Szele E, Gőbel Gy, Szanyi I, Ember I: Az Isoflurane hatása apoptotikus jelátviteli gének expressziójára. Népegészségügyi Tudományos Társaság XVII. Nemzetközi Kongresszusa, 2009. április 17-18., Marosvásárhely Hungarian presentations: 1. Szele E, Gombos K, Ember I: Biodízel előállítás során nyert tisztított glicerin frakció apoptotikus génekre gyakorolt hatásának vizsgálata állatkísérletes modellben. Fiatal Higiénikusos V. Fóruma, 2009. május 1416., Eger 2. Szele E, Gombos K, Ember I: Biodízel előállítás során nyert különböző tisztaságú glicerin frakciók apoptotikus génekre gyakorolt hatásának összehasonlítása állatkísérletes modellben. Magyar Higiénikusok Társasága XXXIX, 2009. október 6-8., Balatonvilágos 3. Szele E, Gombos K, Ember I: Biodízel előállítás során visszamaradt glicerin frakció hatásának vizsgálata a metabolizmusban kulcsszerepet játszó citokróm gének kifejeződésére. Magyar Higiénikusok Társasága IX. Nemzeti Kongresszusa, 2010. október 5-7., Balatonvilágos
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Szele E, Gombos K, Ember I: A biodízel-glicerin hatásának vizsgálata jelátviteli folyamatokban és metabolizmusban kulcsszerepet játszó gének kifejeződésére CBA/CA egerekben. A glicerin takarmányozási hasznosítása, 2011. május 6., Mosonmagyaróvár 5. Szele E, Gombos K, Juhász K, Wolher V, Kovács A, Ember I: Biodízelgyártás során keletkezett anyagok hatása a sejttúlélésben kulcsszerepet játszó microRNS-ek és messenger RNS-ek kifejeződésére BALB/c egerekkel végzett rövidtávú állatkísérletes modellben. Fiatal Higiénikusok VI. Fóruma, 2012. május 10-11., Esztergom 6. Szele E, Gombos K, Juhász K, Wolher V, Ember I: Biodízelgyártás során melléktermékként keletkezett „szappanos vízzel” kezelt talajon termesztett búza hatásának vizsgálata daganatképződésben és metabolizmusban kulcsszerepet játszó miRNS-ek és mRNS-ek génexpressziójának mérésével. Magyar Higiénikusok Társasága XLI. vándorgyűlése, 2012. október 3-5., Esztergom 7. Gombos K, Szele E, Varjas T, Puskás L, Kozma L, Juhász F, Ember I:, Budapest: Microarray módszer alkalmazása pajzsmirigydaganatok korai felismerésében. Magyar Onkológusok Társaságának XXVII. Jubileumi Kongresszusa, 2007. november 8-10., Budapest 8. Gombos K, Szele E, Varjas T, Puskás L, Kozma L, Juhász F, Ember I: Microarray módszer alkalmazása pajzsmirigy daganatok korai felismerésében. Fiatal Higiénikusok Fóruma, 2008. május 29-31., Győr 9. Gőbel Gy, Gerlinger I, Pytel J, Szanyi I, Szele E, Gombos K, Ember I: A Malignus nyálmirigy daganatok retrospektív vizsgálata az 1986-2006-os időtartamban. Magyar Epidemiológiai Társaság IV. Nemzetközi Kongresszusa, 2008. november 25-26., Pécs International posters: 1. Szele E, Gombos K, Kovács A, Ember I: Feeding purificated glycerol from Biodiesel to CBA/CA mice: effects on single strand DNA damage inducible GADD45α and NFKB expressions. Eighth International Conference of Anticancer Research, 2008. október 17-22., Kos, Görögország 2. Ember I, Gombos K, Varjas T, Kiss I, Szele E, Puskás L, Kozma L, Juhász F, Varga Zs, Ember Á: Characterization of microarray gene expression profiles of thyroid tumours. Dubai International Oncology Conference, 2007. február 19-27., Al Ain, Egyesült Arab Emirségek 3. Kádár B, Gombos K, Szele E, Gőbel Gy, Szanyi I, Ember I: Effects of Isoflurane on NFKB1, GADD45α JNK1 expressions in the vital organs of CBA/CA mice. Eighth International Conference of Anticancer Research, 2008. október 17-22., Kos, Görögország 4. Gombos K, Szele E, Puskás L,Kozma L, Juhász F, Gőbel Gy, Szanyi I, Ember I: Analysis of the connections between signal transduction mechanisms in early stage thyroid tumours. Eighth International Conference of Anticancer Research, 2008. október 17-22,. Kos, Görögország 5. Gőbel Gy, Gerlinger I, Pytel J, Szanyi I, Szele E, Gombos K, Ember I: Malignus nyálmirigy daganatok epidemiológiai sajátosságai. Népegészségügyi Tudományos Társaság XVII. Nemzetközi Kongresszusa, 2009. április 17-18., Marosvásárhely Hungarian posters: 1. Szele E, Gombos K, Varjas T, Puskás L, Kozma L, Juhász F, Ember I: Microarray módszer alkalmazása a pajzsmirigy daganatok korai felismerésében. Népegészségügyi Tudományos Társaság XVI. Nemzetközi Kongresszusa, 2008. április 17-19., Pécs 2. Szele E, Gombos K, Ember I: Az SZME2 biodízel előállítás során nyert tisztított glicerin frakció NFKB, JNK és GADD45A génekre gyakorolt hatásának vizsgálata állatkísérletes modellben. Magyar Epidemiológiai Társaság IV. Nemzetközi Kongresszusa, 2008. november 28-29., Pécs 3. Szele E, Gombos K, Kovács A, Ember I: Biodízel-glicerin hatása a sejttúlélésben kulcsszerepet játszó microRNS-ek és messenger RNS-ek kifejeződésére BALB/c egerekkel végzett rövidtávú állatkísérletes modellben. Magyar Epidemiológiai Társaság VI. Nemzetközi Kongresszusa, 2011. november 25-26., Pécs 4. Gombos K, Szele E, Herczeg M,. Brunner Zs, Szanyi I, Molnár K, Gergely P, Mucsi Gy, Varga Zs, Ember I: Effects of VitaCalen consumption on the survival of CBA/CA mice. Magyar Molekuláris és Prediktív Epidemiológiai Társaság Kongresszusa, 2006. november 3-5., Pécs 5. Gombos K, Szele E, Varjas T, Puskás L, Kozma L, Juhász F, Ember I: Jelátviteli mechanizmusok összefüggéseinek vizsgálata pajzsmirigy daganatokban. Kertai emlékülés, 2007. június, Pécs 6. Gombos K, Szele E, Varjas T, Puskás L, Kozma L, Juhász F, Ember I: Jelátviteli mechanizmusok összefüggéseinek vizsgálata pajzsmirigy daganatokban. Népegészségügyi Tudományos Társaság XVI. Nemzetközi Kongresszusa, 2008. április 17-19., Pécs 7. Kádár B, Gombos K, Szele E, Beregi A, Varga Zs, Sebestyén A, Ember I: Az izoflurán onko- és tumorszuppresszor génekre kifejtett hatásának vizsgálata CBA/Ca egerekben. Népegészségügyi Tudományos Társaság XVI. Nemzetközi Kongresszusa, 2008. április 17-19., Pécs 8. Kádár B, Gombos K, Szele E, Gőbel Gy, Szanyi I, Ember I: Az Isoflurane hatása az NFKB1, JNK1 és GADD45α gének expressziós mintázatára. Magyar Epidemiológiai Társaság IV. Nemzetközi Kongresszusa, 2008. november 28-29., Pécs
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Gőbel Gy, Gerlinger I, Pytel J, Szanyi I, Szele E, Gombos K, Ember I: A Malignus nyálmirigy daganatok retrospektív vizsgálata az 1986-2006-os időtartamban. Magyar Epidemiológiai Társaság IV. Nemzetközi Kongresszusa, 2008. november 28-29., Pécs 10. Gombos K, Szele E, Gőbel Gy, Puskás L, Kozma L, Juhász F, Ember I: Pajzsmirigy daganatok génexpressziós profiljának meghatározása cDNS microarray módszerrel. Magyar Epidemiológiai Társaság IV. Nemzetközi Kongresszusa, 2008. november 28-29., Pécs
VIII. Funding In order to carry out studies on biodiesel, the Institute of Public Health at the University of Pécs, Medical Faculty as part of a consortium announced a tender of the National Office for Research and Technology called “Jedlik Ányos Program” and received support on two occasions. („Technológia és állati takarmány kompozíciók biodízel G-fázis melléktermék hasznosításával”, ID: NFKP07-aAGROÖK07, „Fenntartható biodízel technológia és hozzáadott értékű melléktermékek”, ID: TECH-09-A4-2009-0133, BDREVAM2.
IX. Acknowledgement My interest in primary prevention, cancer research and related molecular epidemiology is thanks to Prof. István Ember whose huge professional experience, dedication and personality has deeply influenced me. As a student of Prof. István Ember, I acquired all the skills related to my thesis. He followed my scientific work from the first step, and his advice and constructive criticism helped my studies. I would like to say thank you to my supervisor Prof. Dr. István Kiss for his teaching and professional support. I would like to say thank you to Dr. Andras Kovacs for the management and the availability of sample materials. I would like to say thank you to Dr. Katalin Gombos for participating all of my studies and professional support. I would like to say thank you to all of those colleagues in the Institute of Public Health who helped in the laboratory work or gave me any administrative help. I would like to express my thanks to my family; with their love, patience and providing gave me the possibility to work on my thesis.
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